DE102005046753A1 - Microscopy and microscope - Google Patents

Abstract

A microscopy method is provided for generating an image of an image field lying in a predetermined depth of a sample to be examined, with a plurality of illuminating fonts, in each of which a part of the image field is illuminated with a focused illuminating beam which, due to an interaction with the sample, generates sample radiation causes detection steps in which the generated sample radiation is detected, and an evaluation step in which the image is generated on the basis of the detected sample radiation, a first and a second detection step being carried out during each illumination step, with the first detection step being in focus and outside of it Focus generated sample radiation and in the second detection step a smaller portion of the sample radiation generated in the focus than in the first detection step and outside the focus generated sample radiation is detected, and in the evaluation step d he sample radiation detected in the second detection step is used to reduce the non-focal portion of the sample radiation detected in the first detection step.

Description

The The invention relates to a microscopy method for producing a Image of one at a predetermined depth of one to be examined Sample lying field of view, with several lighting steps, in each of which a part of the image field with a focused Illumination beam illuminated due to an interaction with the sample the generation of sample radiation causes detection steps, in which the generated sample radiation is detected, and a Evaluation step, in which on the basis of the detected sample radiation the Image is generated. Furthermore, the invention relates to a microscope for Generating an image of one at a predetermined depth field, with a lighting module, the illuminated in several lighting steps, the image field, wherein in each illumination step in each case a part of the image field with a focused illumination beam is illuminated due to an interaction with the sample the generation of sample radiation causes a detection module that detects generated sample radiation, and an evaluation module based on the detected sample radiation the picture is generated.

Around the desired to achieve confocal deep discrimination, so that the image of the lying in a predetermined depth of the sample to be examined Image field can be generated by laser scanning microscopy a diaphragm is used, which shadows unwanted sample light.

It is also known that the Achieving Confocal Deep Discrimination in the Far Field with partial illumination of the image field (eg a line illumination) structuring or intensity modulation) of the illumination can be used. By a phase shift of the structured Lighting can then be a deeply discriminated optical cut calculated and so the desired Image of the object to be generated. As in M.A.A. Neil et al. "Method of obtaining optical sectioning by using structured light in a conventional microscope "Optics Letters 22 (24) 1997, 1905-1907 This can be achieved with three phase images at 0 °, 120 ° and 240 ° become.

to Structuring of the illumination will be either grating in the illumination beam path, the interference of coherent Partial beams or the use of diffractive optical elements proposed. A disadvantage is the resulting inflexibility and the increased Effort, as with a change of the lens of the laser microscope In general, a change of structuring is required. This must then In general, another grating will be provided, the interference the coherent partial beams changed be used or another diffractive optical element become.

outgoing It is the object of the invention to provide a microscopy method and a microscope of the type mentioned in such a way that the generation deeply discriminated optical sections is easily possible, without physical diaphragms for shadowing of extra-focal sample radiation to have to provide.

According to the invention Task in a microscopy method of the type mentioned solved by that during each Illumination step, a first and a second detection step carried out being in focus as well as outside in the first detection step the focus generated sample radiation and in the second detection step a smaller proportion of the sample radiation generated in the focus than generated in the first detection step and out of focus Sample radiation is detected, and that in the evaluation step in the second Detection step detected sample radiation is used to the sample radiation detected in the first detection step the of-focus Reduce the proportion.

It is thus the present due to the focus spatial Limited lighting within the image field used to in the first and second detection steps at substantially the same Sample radiation outside the focus is generated, different proportions of the focus generated To detect sample radiation. This will then be in the evaluation step used to advantage, the non-focal portion of the first Detection step to reduce detected sample radiation.

Consequently can produce a deeply discriminated cut with little effort without a physical shutter to mask the out-of-focus To provide sample radiation.

There a provision of a physical aperture is not necessary and because of the focus given a spatial limitation of the lighting is, one gains on the detection side a spatial degree of freedom. If the illumination beam z. B. punctiform is focused, you can spectrally split the detected sample radiation and by means of z. B. detect a line detector spectrally resolved. The additional Degree of freedom (or the additional spatial Coordinate) can thus be used in the detection steps spectrally detect and at the same time the desired deeply discriminated Generating the image of the image field to realize.

There none the extra-focs Sample radiation shielding aperture is necessary, a greater detection efficiency be achieved because much more sample light (compared to a method with pinhole aperture) and for the evaluation can be used.

Especially can not generate any of the focus in the second detection step Sample radiation and thus generated only out of focus sample radiation be detected. The proportion of the sample radiation generated in the focus, which is detected in the second detection step is thus zero.

The both detection steps can while at least one illumination step are performed simultaneously. This can be the measuring time as possible keep low.

alternative it is also possible that both Detection steps during at least one lighting step are performed sequentially in time. In this case one can for both lighting steps employed a single detector.

Especially in the first detection step, the first section the sample surface and in the second detection step that of a second section the sample surface emerging sample radiation detected, with both sections together borders or the second section covers the first section only partially. The two sections can directly adjoin (touch) or also from each other be spaced. Furthermore, in the first detection step, the Focusing area in the image field on a detector, while in second detection step detects an adjacent area in the image field becomes.

in the Evaluation step, the detected in the second detection step Subtracted signal from the signal detected in the first detection step become. Naturally can be a relative weighting of the two signals to each other considered become.

Especially can the lighting steps, the detection steps and the evaluation step for many Image fields are performed at different predetermined depths of the sample, thus generate several deeply discriminated sectional images of the sample to be able to.

from that can then with known methods and three-dimensional sample images be generated.

at the method, the illumination beam (preferably diffraction-limited) punctiform or linear be focused.

The Task is further in a microscope of the type mentioned solved by that during each Lighting step, the detection module has a first and a second Performs detection step, being in focus as well as outside in the first detection step the focus generated sample radiation and in the second detection step a smaller proportion of the sample radiation generated in the focus than generated in the first detection step and out of focus Sample radiation is detected, and that the evaluation module in the second detection step uses detected sample radiation to at the sample radiation detected in the first detection step the non-focal Reduce the proportion.

By This type of detection can be used to shadow a physical shutter the non-focal Sample radiation can be dispensed with, as by the spatial Limit of the focused in the field of illumination beam present local Modulation of the illumination by means of the two detection steps is exploited to different proportions of the generated in the focus To detect sample radiation. In particular, in the second detection step only outside the focus generated sample radiation can be detected. The amount the sample radiation generated in the focus, in the second detection step is detected, then is zero.

Further you can spectral in the two detection steps, the sample radiation disbanded detect. It is exploited that no physical aperture for shading the extra-focial Proportion of the sample radiation is necessary and that by the spatial Limitedness of the focused illumination beam on the detection side another degree of freedom is present. So can be focused at a point Illumination beam spectrally split the sample radiation and by means of a line detector be detected spectrally. The spectral splitting takes place in Direction of the extension direction of the line detector. Of course you can as a line detector z. For example, only one row or one column of a spatially resolving Surface detector used become. When the illumination beam focuses in a line becomes, becomes an area detector used, in which case the spectral splitting preferably transverse to the extension direction of the linear focus. The spectral Splitting can be performed with any suitable optical element (with appropriate Dispersion) be, for. B. by means of a prism or a diffraction grating. The Detection module can thus optics for spectral splitting of Sample radiation and at least one spectrally split Sample radiation spectrally resolved have detecting detector.

The Detection module can include two detectors, creating a simultaneous Carrying out the two detection steps over both detectors possible is. Alternatively, the detection module may also comprise a single detector so that both Dektionschritte be carried out temporally successively.

in the The first detection step may consist of a first section of the sample surface and in the second detection step that of a second section the sample surface emerging sample radiation are detected, the two Sections of the sample surface adjacent (directly or spaced apart) or the second section only partially covers the first section.

Further the detection module can be designed such that the focus area imaged in the image field in the first detection step on a detector is and adjacent to the focus area in the second detection step area of the image field is imaged onto a detector of the detection module.

The Evaluation module can the detected in the second detection step Subtract signal from the signal detected in the first detection step, wherein a weighting of the two signals to each other is possible. This will be easier Way of the extra-focal Reduced proportion in the detected signal of the first detection step.

The Lighting module may include a scanner module, the illumination beam so distracts that entire image field is illuminated.

Further For example, the lighting module may use the lighting beam as point or line Focus focused illumination beam on the image field.

The The invention will be described by way of example with reference to the accompanying drawings explained in more detail. It demonstrate:

1 a schematic view of the microscope according to the invention;

2 an enlarged view of the detection module of 1 ;

3 a cross-sectional view of the sample to be examined;

4 a plan view of the detectors of 2 ;

5 a modification of the detectors of 4 ;

6 an alternative embodiment of the Dektionsmoduls of 1 , and

7 and 8th a further alternative embodiment of the detection module of 1 ,

At the in 1 In the embodiment shown, the microscope is designed as a laser scanning microscope, which is a light source module 1 , a scan module 2 , a lens 3 , a recording module 4 as well as an evaluation module 5 includes.

The light source module 1 that has a laser 8th and a beam shaping optics 9 generates, generates a laser beam LS1, the one between the light source module 1 and the scan module 2 switched beam splitter 6 to the scan module 2 is directed, the beam LS1 so over the sample 7 deflects a field of view within the sample is completely illuminated. The laser beam LS1 is thereby by means of the lens 3 into the sample 7 focused, in the depth of the image field from which an image is to be generated (optical section).

In the embodiment described here, the laser beam LS1 is focused in a punk shape (preferably diffraction-limited), so that the scan module 2 The laser beam LS1 deflects in two independent directions from each other, thus the entire image field within the sample 7 with the focused laser beam LS1 to sweep.

Due to the interaction of the laser beam LS1 with the sample, sample radiation LS2 is generated, which is transmitted via the objective 3 on the scan module 2 that meets the sample 7 descending sample radiation LS2, so that the sample radiation LS2 behind the scan module 2 is present as stationary beam LS2. The beam splitter 6 is formed so that it transmits the sample radiation LS2, so that these on the detection module 4 meets.

The generated sample radiation can, for. B. fluorescent light, luminescent light, be reflected, transmitted and / or scattered light.

As in 2 is shown schematically comprises the detection module 4 a detection optics 11 and a first and second detector D1, D2, whose signals are the evaluation module 5 be supplied. The focus area of the image field (ie the area in which the laser beam LS1 focuses) is imaged onto the detector D1, while an area adjacent thereto is imaged onto the detector D2.

This will be in conjunction with 3 explained in more detail. 3 shows a cross section through the sample to be examined 7 , where the beam waist 12 of the illumination beam LS1 is shown. Between the dashed lines horizontal lines L1 and L2 is the displayed depth range (image field) of the sample 7 , In this area, the laser beam LS1 is focused. This can be seen from the fact that the beam waist 12 has the smallest diameter in this area.

By the image of the focus area on the detector D1 arrives the detector in the oblique hatched area B1 generated sample radiation. One recognises, that beside the desired confocal Sample radiation from the section of area B1 between the dashed Lines L1 and L2 are still the extra-focal Sample radiation passes, above and below the line L1 and L2 is generated in the area B1.

On the detector D2 enters the horizontally hatched area B2 generated sample radiation. Because with the detector D2 only one area next to the focus area is detected, applies to the detector D2 only the sample radiation outside the focal area (between L1 and L2) of the beam LS1 within of the area B2 is generated. In the example described here sees the detector D2 thus generated only out of focus Sample radiation LS2.

In 4 For clarity, the detector-side illumination distribution is shown in the focus. The focused laser beam LS1 is imaged only on the detector D1 (circle F1), so that the detector D2 does not receive any confocal signals.

Assuming that the two detectors D1 and D2 have approximately the same area and sensitivity, the confocal signal Sc and the non-confocal signal Snc may be given as follows: Sc = S1 - n · S2 and Snc = S1 + n * S2 - Sc = 2 * n * S2 where S1 and S2 are the signals of the detectors D1 and D2. The constant n can be empirically z. B. from the minimization of the background signal during the measurement. Typical values for n are 1 to 1, 3.

It has been shown that in particular with thick samples the exact position and the sharpness of the dividing line between both detectors with respect to the diffraction-limited focus spot the focal area is not essential.

It is generally convenient to arrange the detectors so that the boundary between both detectors D1 and D2 is about 1 to 2 Airy Unit radii away from the center of the spot distribution on the detector D1. The Airy unit is defined on the object side as 1 AU = 1.22 λ / NA, where λ is the vacuum wavelength of the illumination beam LS1 and NA is the numerical aperture of the objective 3 is. Also, a spacing of the two areas B1 and B2, as in 3 is shown for simplified graphical representation, for example, less than 1AU between two detectors D1 and D2 (for example, by a web) is not a problem.

In 5 the detectors D1 and D2 are shown in the case that there is no punctiform focusing, but a line-shaped focusing. In the same way, the linear focus (ellipse F2) is then imaged onto the detector D1, as in FIG 5 is shown. Of course, then the scan module 2 adapted accordingly, so that under certain circumstances (if the line is so long that it covers the entire image field in the line direction) only a deflection transverse to the extension direction of the linear focus is necessary.

For the control of the microscope and the implementation of the described steps, the microscope has a control unit 10 on.

Of course, one is not limited to a point or line focusing. It is essential that the focusing has a local boundary in at least one direction in the image field, wherein this limit is preferably as sharp as possible. The steepness of the boundary should preferably be at least one spatial frequency at half the cutoff frequency of the objective 3 correspond.

In 6 is an alternative embodiment of the detection module 4 shown. This is where the detection module points 4 a beam splitter 13 on, for example, each reflects half of the incident sample light LS2 and transmits and thus split into two detection arms. In the right to the right detection arm in 6 both the focal component LS2c and the non-focal component LS2nc are plotted, with only the confocal component LS2c shown in the upward-pointing detection arm for the sake of simplicity. Due to the local arrangement of the detectors D1, D2 in the detection arms, only the non-focal component LS2nc strikes the detector D2, while the focal component LS2c strikes the detector D1.

In 7 and 8th is an embodiment of the detection module 4 shown, in which only a single detector D1 is provided. Between the detection optics 10 and the detector D1 is a rotatable glass plate 14 arranged, which ensures, depending on the rotational position, that either the focal component LS2c or the non-focal component LS2nc on the active surface of the detector 1 meets.

When the illumination beam or the focused laser beam LS1 is focused in a point-like manner, as in connection with 1 described, the line detectors of 5 be used for spectrally resolved detection. For this purpose, the detected sample radiation must be split only spectrally in the direction of extension of the line detector before it hits the line detectors D1, D2.

The same applies when the laser beam LS1 is focused linear. Then is at least one spatial resolution flat Detector necessary, with z. B. in the column direction spatially resolved line focus and in the row direction of the spectral resolution in this direction line focus spectrally resolved is detected.

Claims (16)

A microscopy method for producing an image of an image field lying at a predetermined depth of a sample to be examined, comprising a plurality of illumination steps, in each of which a part of the image field is illuminated with a focused illumination beam that causes the generation of sample radiation due to an interaction with the sample, detection steps, in which the generated sample radiation is detected, and an evaluation step in which the image is generated on the basis of the detected sample radiation, characterized in that during each illumination step, a first and a second detection step are performed, wherein in the first detection step in focus and out of focus generated sample radiation and in the second detection step, a smaller proportion of the sample radiation generated in the focus is detected as in the first detection step and outside the focus generated sample radiation, and that in the evaluation step, the i In the second detection step, detected sample radiation is used to reduce the extra-focal fraction in the sample radiation detected in the first detection step. The method of claim 1, wherein the two detection steps while at least one illumination step are performed simultaneously. The method of claim 1, wherein the two detection steps while at least one lighting step are performed sequentially in time. Method according to one of the above claims, wherein in the first detection step, that of a first section of the sample surface occurring sample radiation and in the second detection step the off Sample radiation emerging from a second section of the sample surface is detected, with both sections adjacent to each other or the second section covers the first section only partially. Method according to one of the above claims, wherein in the evaluation step that detected in the second detection step Subtracted signal from the signal detected in the first detection step becomes. Method according to one of the above claims, wherein the lighting steps, the two detection steps and the evaluation step for several image fields in different predetermined depths of Sample performed become several deeply discriminated sectional images of the sample to create. Method according to one of the above claims, wherein the lighting beam point or line is focused. Method according to one of the above claims, wherein in the two detection steps, the sample radiation spectrally disbanded is detected. Microscope for generating an image of one in one predetermined depth of a sample to be examined lying image field, With a lighting module in several lighting steps illuminated the image field, wherein in each lighting step respectively a part of the image field is illuminated with a focused illumination beam This is due to an interaction with the sample generation of sample radiation causes a detection module that generated Sample radiation detected, and an evaluation module, the on Based on the detected sample radiation generates the image, thereby characterized in that during each Lighting step, the detection module has a first and a second Performs detection step, in which generated in the first detection step in focus and out of focus Sample radiation and in the second detection step a lesser Proportion of the sample radiation generated in the focus as in the first detection step as well as outside the focus generated sample radiation is detected, and that the evaluation module uses the sample radiation detected in the second detection step, at the sample radiation detected in the first detection step the non-focal To reduce the share. Microscope according to claim 9, wherein the detection module includes two detectors, so that a simultaneous execution the two detection steps possible is. Microscope according to claim 9, wherein the detection module for the both detection steps has a single detector. Microscope according to one of claims 9 to 11, wherein in the first Detection step from a first section of the sample surface and in the second detection step from a second section of the sample surface emerging sample radiation is detected, the two sections the sample surface contiguous or the second section the first section only partially covered. Microscope according to one of claims 9 to 12, wherein the evaluation module the detected in the second detection step from the signal in the first Detection step detected signal subtracted. Microscope according to one of claims 9 to 13, wherein the illumination module a scanner module, which illuminates the illumination beam over the Image field directs. Microscope according to one of claims 9 to 14, wherein the illumination module illuminated the image field with a point or line focused illumination beam. Microscope according to one of claims 9 to 15, in which two detection steps, the sample radiation detected spectrally resolved becomes.